The ubiquitin-proteasome system (UPS), which controls the degradation of proteins inside the cell, plays an integral role in maintaining cellula protein homeostasis. Dysregulation of the UPS is associated with a growing number of diseases including cancers, neurodegenerative disorders, and viral and bacterial infections. As a result, regulators of the UPS have received considerable attention for developing new human therapeutics. The current FDA approved therapeutics, which target the UPS, are efficacious but also fairly toxic - probably because they completely shut down the activity of the UPS. This proposal seeks to address this problem by developing small molecule inhibitors of DCN1 that regulate the UPS without completely blocking its activity. DCN1 is a recently identified protein whose dysregulation is associated with a number of human cancers. DCN1 activity is controlled by its interaction with UBC12. Therefore, the goal of this proposal is to develop potent and selective small molecule inhibitors that disrupt the DCN1-UBC12 protein-protein interaction and use them to elucidate the biological effects of DCN1 mediated UPS regulation. Aim 1 addresses the optimization of DCN1-UBC12 interaction inhibitors, identified by a high-throughput screening campaign, based on biochemical potency and models for cellular and organismal bioavailability. Aim 2 addresses whether or not chemical disruption of the DCN1-UBC12 interaction functionally and selectively inhibits neddylation, cullin-RING E3 ligase activity, UPS activity, and effectively suppresses tumor growth in clinically relevant in vivo models. If the DCN1-UBC12 interaction is validated as a target for regulation of the UPS, this new mechanism of action will provide a useful tool for elucidating the molecular mechanisms of DCN1 mediated CRL activity and regulation of the UPS. These studies will increase our understanding of a key life process and potentially lay the foundation for significant advances in the development of highly selective therapeutics.